Chemistry & Biology

Article

Amide N-Glycosylation by Asm25,an N-Glycosyltransferase of AnsamitocinsPeiji Zhao,1,5 Linquan Bai,2,5,* Juan Ma,1 Ying Zeng,1 Lei Li,2 Yirong Zhang,2 Chunhua Lu,3 Huanqin Dai,1

Zhaoxian Wu,3 Yaoyao Li,3 Xuan Wu,3 Gang Chen,1 Xiaojiang Hao,1 Yuemao Shen,1,3,* Zixin Deng,2

and Heinz G. Floss4

1State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,Kunming, Yunnan 650204, China2Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China3Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen,

Fujian 361005, China4Department of Chemistry, Box 351700, University of Washington, Seattle, WA 98195-1700, USA5These authors contributed equally to this work.

*Correspondence: bailq@sjtu.edu.cn (L.B.), yshen@xmu.edu.cn (Y.S.)

DOI 10.1016/j.chembiol.2008.06.007

SUMMARY

Ansamitocins are potent antitumor maytansin-oids produced by Actinosynnema pretiosum. Theirbiosynthesis involves the initial assembly of a macro-lactam polyketide, followed by a series of postpoly-ketide synthase (PKS) modifications. Three ansami-tocin glycosides were isolated from A. pretiosumand fully characterized structurally as novel ansami-tocin derivatives, carrying a b-D-glucosyl groupattached to the macrolactam amide nitrogen in placeof the N-methyl group. By gene inactivation andcomplementation, asm25 was identified as the N-gly-cosyltransferase gene responsible for the macrolac-tam amide N-glycosylation of ansamitocins. Soluble,enzymatically active Asm25 protein was obtainedfrom asm25-expressing E. coli by solubilizationfrom inclusion bodies. Its optimal reaction condi-tions, including temperature, pH, metal ion require-ment, and Km/Kcat, were determined. Asm25 alsoshowed broad substrate specificity toward otheransamycins and synthetic indolin-2-ones. To thebest of our knowledge, this represents the firstin vitro characterization of a purified antibiotic N-gly-cosyltransferase.

INTRODUCTION

Ansamitocins are maytansinoids of microbial origin, produced

by Actinosynnema pretiosum ssp. auranticum ATCC 31565.

They differ structurally from their higher-plant congeners, such

as the parent compound maytansine, only by carrying simpler

acyl groups at C-3 (Figure 1A; Higashide et al., 1977). Like the

plant maytansinoids, ansamitocins show extremely potent

cytotoxicity against various tumor cells (Ootsu et al., 1980).

Structurally, the maytansinoids belong to the ansamycin family

of polyketide macrolactams (Rinehart and Shield, 1976), exem-

plified by the antitubercular agent rifamycin and the antitumor

Chemistry & Biology 15, 8

agent geldanamycin. Their molecular architecture is character-

ized by an aromatic chromophore with an aliphatic chain (ansa

chain) connected back to a nonadjacent position through an

amide linkage. Despite carrying either benzenic (ansamitocins,

geldanamycin) or naphthalenic (rifamycin, naphthomycin) chro-

mophores, genetic and feeding experiments have revealed that

all the ansamycins share the same polyketide starter unit,

3-amino-5-hydroxybenzoic acid (AHBA) (Ghisalba and Nuesch,

1981; Hatano et al., 1982; Kibby et al., 1980). The ansamitocin

biosynthetic genes were cloned from a cosmid library of A. pre-

tiosum genomic DNA using the AHBA synthase gene rifK (Kim

et al., 1998) from the rifamycin biosynthetic gene cluster as

probe (Yu et al., 2002). Notably, two AHBA synthase genes

were found, and sequencing of the surrounding DNA localized

the genes expected to be required for ansamitocin biosynthesis

in two clusters separated by 30 kb of nonessential DNA

(Figure 1B). Bioinformatic analysis, gene inactivation, and ex-

pression experiments demonstrated that cluster I contains

most of the ansamitocin biosynthetic genes, whereas cluster II

carries only four of the seven genes required for AHBA formation

(Figure 1B; Yu et al., 2002). AsmA-D are four large open-reading

frames (ORFs) encoding the loading domain and seven chain-

elongation modules of a type I multifunctional polyketide syn-

thase (PKS). Gene asm9, located immediately downstream of

asmD, encodes the amide synthase responsible for macrolac-

tamization of the nascent polyketide chain and release of proan-

samitocin from the PKS (Kato et al., 2002; Yu et al., 2002).

As in other microbial secondary metabolites, post-PKS modi-

fications are important for the biological activity of the ansamito-

cins (Cassady et al., 2004). Six modifying reactions are required

to turn proansamitocin into ansamitocins, and cluster I contains

candidate genes for each of these. Their functions and the order

in which they act were determined by in vivo gene inactivation,

feeding experiments with accumulated compounds, and

in vitro enzymatic analysis of heterologously expressed gene

products. This defined a predominant pathway from proansami-

tocin to ansamitocins, consisting of chlorination (Asm12), carba-

moylation (Asm21), O-methylation (Asm7), acylation (Asm19),

epoxidation (Asm11), and N-methylation (Asm10) (Moss et al.,

2002; Spiteller et al., 2003). Notably, the only ansamitocin prod-

ucts of the asm10 deletion mutant are macrolactam amide

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Chemistry & Biology

Amide N-Glycosylation

Figure 1. The Post-PKS Modifications of N-Desmethylansamitocins and the Ansamitocin Biosynthetic Gene Cluster

(A) Chemical structures of ansamitocins (APs), N-desmethylansamitocins (PNDs), and ansamitocinosides (AGPs). The putative scheme for the conversion of

PNDs to APs and AGPs by Asm10 and Asm25, respectively.

(B) The ansamitocin biosynthetic gene cluster. The N-glycosyltransferase gene, asm25, and the N-methyltransferase gene, asm10, are indicated by a circle in

Cluster I. AHBA: 3-amino-5-hydroxybenzoic acid; MM-ACP: methoxy-malonyl-ACP.

N-desmethylansamitocins (PNDs) (Figure 1A), indicating that

N-methylation is the terminal step in the post-PKS modifications

of ansamitocin biosynthesis (Spiteller et al., 2003).

Structural variations of the ansamitocins reside mainly in the

C-3 acyl moiety. Other minor components with hydroxyl func-

tions at C-15 or the C-14 methyl group, or lacking the N-methyl

group, were also found in A. pretiosum (Izawa et al., 1981).

Recently, from the cultures of A. pretiosum on solid ISP2 me-

dium, we have isolated a series of novel N-glycosylated ansami-

tocins with a b-D-glucosyl residue attached to the macrolactam

amide nitrogen instead of the N-methyl group (Figure 1A). Ansa-

mitocinosides P-1 and P-2 (AGP-1, AGP-2), the main compo-

nents, were fully characterized structurally (Lu et al., 2004; Ma

et al., 2007). In the present work, we show through gene inacti-

vation and complementation the gene encoding the unique am-

ide N-glycosyltransferase (N-Gtf) to be asm25, and we report the

overexpression, purification, and characterization of Asm25.

Soluble active Asm25 protein was purified from inclusion bodies

formed in asm25-expressing E. coli, which is unprecedented in

studies of antibiotic N-Gtfs. Additionally, the substrate range of

864 Chemistry & Biology 15, 863–874, August 25, 2008 ª2008 Elsev

Asm25 was probed with other ansamycins and synthetic lac-

tams (indolin-2-one derivatives).

RESULTS

Isolation and Structure Elucidationof Ansamitocinoside P-3Previously, we reported the isolation of two unique ansamitocin

N-glycosides, macrolactam amide N-desmethyl-N-b-D-gluco-

pyranosylansamitocins P-1 and P-2, named as ansamitocino-

sides P-1 (AGP-1) and P-2 (AGP-2) from A. pretiosum ATCC

31565 (Lu et al., 2004; Ma et al., 2007), and detected additional

ansamitocinosides by LC-MS. In the present work, one more

glycoside of a macrolactam amide N-desmethylansamitocin,

namely ansamitocinoside P-3 (AGP-3), was isolated from the

ethyl acetate extract of A. pretiosum cultivated on solid ISP2 me-

dium by improved preparative TLC methods (Ma et al., 2007).

The molecular formula of AGP-3 was determined to be

C37H51N2O14ClNa (m/z 805.2917 [M + Na]+, calculated:

805.2926) based on HR-ESI-MS data. The 13C-NMR and DEPT

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Chemistry & Biology

Amide N-Glycosylation

Figure 2. Inactivation of asm25 and Complementation of the Mutant

(A) Schematic representation of the 465 bp deletion within asm25 from the A. pretiosum genome. In shuttle plasmid pJTU478, a truncated asm25 was generated

by linking the 2.7 kb and 3.4 kb genomic fragments originally flanking the deleted 465 bp region. S, Sacl; P, Pstl.

(B) PCR analysis of wild-type A. pretiosum and mutant BLQ11. Whereas wild-type A. pretiosum gave a 1.60 kb PCR-amplified product, mutant BLQ11 yielded

a 1.10 kb product as expected.

(C) ESI-MS profiles of the ansamitocin products of the wild-type, asm25 deletion mutant BLQ11, and asm25-complemented strain BLQ15 cultured on solid ISP2

medium.

spectra of AGP-3 show 37 carbon signals, including 7 methyls, 4

methylenes, 16 methines, and 10 quaternary carbons (see Table

S1A available online). But in the 1H-NMR spectra, the proton sig-

nal attributed to CH3N at d 3.18�3.22 in ansamitocin P-3 (AP-3)

(Kupchan et al., 1977) was missing in AGP-3, indicating the ab-

sence of the N-methyl group in AGP-3. In addition, one more

six-carbon unit (d 85.3, 71.6, 80.0, 71.5, 79.5, 63.3) was assigned

to be a hexosyl moiety. The sugar was determined to be a b-D-

glucopyranosyl moiety based on the unambiguous NMR assign-

ments. Particularly, the JH-1’’,-2’’ = 9.4 Hz indicated a b-glucosidic

linkage. The HMBC correlations between the anomeric proton

at d 5.55 (H-100) and the carbons at d 172.6 (C-1) and d 138.3 (C-18)

revealed the linkage of the glucose unit to the aglycone, N-des-

methylansamitocin P-3 (PND-3), via the macrolactam amide

nitrogen. Therefore, AGP-3 was determined to be N-des-

methyl-N-b-D-glucopyranosylansamitocin P-3 (Figure 1A).

asm25 Is Involved in the Formationof AnsamitocinosidesEarly work had established methylation of the macrolactam am-

ide nitrogen as the terminal step in post-PKS modifications and

identified PND-3 as the last intermediate in ansamitocin biosyn-

thesis (Spiteller et al., 2003). The glycosylation of the same amide

Chemistry & Biology 15, 8

nitrogen could be an alternative step to the methylation catalyzed

by the N-methyltransferase Asm10. Sequence analysis of the

asm gene cluster had identified one gene of unknown function,

asm25, with high sequence homology to glycosyltransferase

(Gtf) genes (Walsh et al., 2003), which at the time had been spec-

ulated to be part of a glycosylation/deglycosylation excretion

system (Figure 1B; Yu et al., 2002). To investigate whether

asm25 is responsible for the N-glycosylation of PNDs, this gene

was inactivated through a 465 bp in-frame deletion from 429 bp

to 894 bp (Figure 2A). The resulting mutant strain BLQ11 was

confirmed by PCR amplification using the total genomic DNA

as template. On the electrophoretic gel, BLQ11 gave the 1.1 kb

expected product, whereas the wild-type showed a 1.6 kb prod-

uct (Figure 2B). The extracts of both BLQ11 and wild-type strains

grown on solid ISP2 medium were analyzed by LC-ESI-MS,

monitoring at the quasimolecular ion peaks of ansamitocins

(APs), ansamitocinosides (AGPs), and N-desmethylansamitocins

(PNDs), respectively. The production of AGP-1, AGP-2, AGP-3,

and AGP-4 (at m/z 755.3 [M + H]+, 769.3 [M + H]+, 783.3 [M + H]+,

and 797.3 [M + H]+, respectively) was observed in the wild-type

but abolished in BLQ11 (Figure 2C).

Further support for the involvement of asm25 in the N-glyco-

sylation at the macrolactam amide nitrogen came from

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Amide N-Glycosylation

Figure 3. Properties of the Renatured

Asm25

(A) A representative Coomassie brilliant blue-

stained SDS-PAGE gel loaded with molecular

weight markers (lane 1), renatured Asm25 (lane

2), inclusion bodies (lane 3), soluble protein (lane

4), and cell-free extract (lane 5) fractions.

(B) Temperature optimum for the macrolactam

amide N-glycosylation of PND-3 catalyzed by

Asm25.

(C) Determination of pH optimum for the macrolac-

tam amide N-glycosylation of PND-3 catalyzed by

Asm25. Asm25 was assayed in 50 mM Na-ace-

tate/citric acid buffer ranging from pH 4.0 to 7.0

(open column), and 50 mM Tris-HCl buffer ranging

from pH 7.0 to 9.0 (solid column).

(D) Effect of metal ions on the activity of Asm25.

Asm25 was assayed in the absence (0 mM, green)

or presence of various metal ions at 0.1 (blue), 1.0

(pink), and 10.0 mM (black) concentrations except

for the concentration of Mg2+ used at 10 (green

and blue), 50 (pink), and 100 mM (black). The pro-

ductions of AGP-3 were quantitated by LC-MS

with authentic AGP-3 as external standard (see

Supplemental Data).

complementation of mutant BLQ11. An intact copy of asm25

under the control of the PermE* promoter was introduced

into BLQ11 by conjugation using an integrative vector

pJTU813, which generated a derivative named as BLQ15.

LC-MS analysis of the extract of BLQ15 showed a chromato-

gram nearly identical to that of the wild-type; that is, the pro-

duction of AGPs was fully restored by expression of the cloned

asm25 (Figure 2C).

In Vitro N-Glycosylation of PNDs by Asm25To verify that Asm25 indeed catalyzes the glycosylation at the

macrolactam amide nitrogen of ansamitocins, the asm25 gene

was amplified by PCR from the A. pretiosum genome and cloned

into plasmid pRSET-B for heterologous expression. The expres-

sion plasmid pJTU812 was transformed into E. coli BL21(DE3)-

plysE and induced at different temperatures and/or with different

concentrations of isopropyl-b-D-thiogalactopyranoside (IPTG).

Asm25 was successfully overexpressed as an N-terminally

His6-tagged fusion protein as detected by SDS-PAGE. The esti-

mated molecular weight of Asm25 recombinant protein is 45 kDa

from the SDS-PAGE (Figure 3A), and its theoretical molecular

weight is 45679.85 Da.

The yield of recombinant protein was highest when the cells

were grown at 37�C at different concentrations of IPTG, but as

shown in Figure 3A, almost all of it was present as inclusion

bodies. In contrast, cells growing at 16�C or 20�C induced

with 0.8 mM IPTG produced only minute amounts of soluble

Asm25, which showed weak catalytic activity when incubated

with PND-3 and UDP-Glc (data not shown). After many unsuc-

cessful attempts to purify the soluble recombinant Asm25 by

metal ion affinity chromatography, the inclusion bodies were

chosen for the preparation of Asm25. Soluble His6-Asm25 pro-

tein with the best enzymatic activity was obtained from the

inclusion bodies by washing with 2.0 M urea and 0.5% Triton,

solubilization in 8.0 M urea, and finally dialysis against renatur-

ation buffer IV with glycerol and glycine (see Experimental Pro-

866 Chemistry & Biology 15, 863–874, August 25, 2008 ª2008 Elsev

cedures). The solution of Asm25 (1.6 mM) in buffer IV with 30%

glycerol and 1% bovine serum albumin (BSA) was used for the

enzyme assays.

Based on the structures of the ansamitocinosides and the

known pathway of ansamitocin biosynthesis (Spiteller et al.,

2003), it was assumed that PNDs are the aglycone substrates

for Asm25. Using as substrate PND-3 isolated from the asm10

mutant grown on solid ISP2 medium, the enzymatic properties

of Asm25 were examined. The reactions were performed with

10 ml PND-3 (20 mM in DMSO), 10 ml UDP-Glc (40 mM; Sigma,

St. Louis, MO), and 0.1 ml basal reaction solution (1.6 mM

Asm25, 50 mM Tris-HCl [pH 8.0], 30% glycerol, 1% BSA,

0.1 M NaCl, 0.1 M glycine, 0.5 mM EDTA, 1.0 mM DDT, and

10 mM MgCl2). The reaction product was identified as AGP-3

by MS (Figure S2B), NMR, and HPLC comparison (Figures

S2H–M and Table S1B), and quantitated by LC-MS with au-

thentic AGP-3. Based on the reaction temperature profile

from 0�C to 50�C, Asm25 purified from the inclusion bodies

was optimally active at 37�C and exhibited more than 70%

of its maximal activity between 35�C and 42�C (Figure 3B).

The pH profile was determined at 37�C in 50 mM Na-ace-

tate/citric acid buffer (pH 4.0–7.0) and 50 mM Tris-HCl buffer

(pH 7.0–9.0). Asm25 showed optimal activity at pH 7.5 and

more than 70% of maximal activity in the pH range of 6.0–

8.0 (Figure 3C).

Because many enzymes that bind dinucleotide substrates re-

quire divalent, oxophilic cations, such as Mg2+ or Mn2+, we

examined the effects of metal ions on the activity of Asm25.

The reactions were performed in the basal reaction solution con-

taining 1.54 mM PND-3 and 3.08 mM UDP-Glc for 4 h at 37�C.

Enzymatic activity improved with the addition of Li+, Zn2+, or

Mn2+ at a concentration of 0.1 mM. Negligible effects on the cat-

alytic activity were observed as the concentrations of Li+ and

Mn2+ increased to 10 mM. However, the activity was partially in-

hibited by 0.1 mM Hg2+ or Cu2+, and almost completely inhibited

by 1 mM Hg2+, Zn2+, or Cu2+ (Figure 3D).

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Chemistry & Biology

Amide N-Glycosylation

Kinetics of Renatured Asm25 with UDP-Glcand PND-3 as CosubstratesKinetic studies of the reaction catalyzed by the refolded

Asm25 were performed using UDP-Glc as sugar donor and

PND-3 as sugar acceptor. When the initial rate was measured

by varying the concentrations of PND-3 at different fixed con-

centrations of UDP-Glc, a set of intersecting lines were ob-

tained by regression analysis of the data at each UDP-Glc

concentration (Figure 4A). Double reciprocal plots of the initial

rate data are shown in Figure 4A. The lines in the double recip-

rocal plots converged, though not to a single point, implying

that Asm25 obeys a ternary complex mechanism in which

both substrates bind prior to product release. In general, two

basic types of mechanisms, ternary complex and ‘‘ping-

pong’’ mechanism, can be deduced from kinetic data based

on the lines in double reciprocal plots. Lines converging to

a single point indicate a ternary complex mechanism, whereas

parallel lines suggest a ping-pong mechanism. The lines in Fig-

ure 4A do not converge to a single point; however, they show

the tendency to converge, which differs from the parallel pat-

tern. Therefore, a ternary complex mechanism can be con-

cluded for Asm25. This was also seen in the Arabidopsis in-

dole-3-acetic acid Gtf, which has a mechanism similar to

Asm25 (Jackson et al., 2001). The slope replot does not

pass through the origin, indicating that the glycosylation is

not of a rapid equilibrium-ordered mechanism (Figure 4B).

The reaction thus proceeds through a ternary enzyme-sub-

strate complex (Lairson et al., 2008; Marangoni, 2003; Price

and Stevens, 1982). The recombinant Asm25 exhibited

Michaelis-Menten kinetics. The Vmax and Km values were de-

termined by classical initial rate experiments and calculated

from Lineweaver-Burk plots (Price and Stevens, 1982). The

Km for PND-3 and UDP-Glc were 17.8 mM and 87.3 mM,

respectively, with kcat of 1.3 $ 10�2 $ s�1.

Figure 4. Kinetic Characterization of Asm25

(A) Initial reaction rates were determined with

PND-3 at 20, 30, 40, and 50 mM and UDP-Glc at

0.2, 0.5, 0.75, 1.0, and 2.0 mM, respectively.

Mean values of three independent experiments

with SD indicated by error bars.

(B) Secondary plot of intercept versus 1/[PND-3]

and slope versus 1/[PND-3].

Substrate Specificity of Asm25The aglycone specificity of Asm25

was evaluated using six maytansinoids

(see the LC-MS profiles in Figure S1),

including PND-1 to -4, N-desmethyl-

desepoxymaytansinol (DDM) and N-des-

methyl-desepoxyansamitocin P-1 (DDP-

1) to react with UDP-Glc, respectively.

The glycosylated products, AGP-1, -2,

-3, -4, corresponding to the four PNDs,

were detected by LC-MS (Figure 5A,

panels 1–4) and HPLC (Figure S3). By

comparison of the relative signal intensi-

ties of the product ions, LC-MS analysis

indicated that the glycosylation of PND-2

to AGP-2 gave the highest yield. This is consistent with the

competition reaction using equimolar amounts of all four

PNDs, which showed that among these PND-2 was the preferred

substrate of the enzyme (Figure 5A, panel 5). In contrast, no con-

version of DDM to its corresponding glycoside was detected

(data not shown). A glycosylation product of DDP-1 was de-

tected, but the conversion efficiency was extremely low (data

not shown). Therefore, among the substrates tested, PND-2

was the best sugar acceptor for the glycosylation reaction cata-

lyzed by Asm25.

The sugar specificity of Asm25 was probed using PND-3 and

four UDP-sugars, UDP-Glc, UDP-galactose, UDP-N-acetyl-glu-

cosamine and UDP-glucuronic acid, and ADP- and GDP-Glc as

substrates. A glycosylated product could only be detected by

LC-MS in the assay with UDP-Glc as sugar donor (Figure 5B

and Figure S4). Because no glycosylated products were formed

in the other five reactions, UDP-galactose, UDP-N-acetyl-

glucosamine, UDP-glucuronic acid, ADP- or GDP-Glc cannot

replace UDP-Glc in the Asm25-catalyzed glycosylation of

PND-3. Therefore, we conclude that Asm25 catalyzes the

in vitro glycosylation of PNDs at the macrolactam amide nitrogen

using UPD-Glc as the sole sugar donor.

In Vitro Glycosylation of Other Ansamycinsand Synthetic Lactams by Asm25To evaluate the substrate range of Asm25 further, five ansamy-

cins—geldanamycin-derived lebstatin and 17-O-desmethylleb-

statin (17-DMLB) (Figure 6; Stead et al., 2000), rifamycin A (Sensi

et al., 1959), rifampicin (Morisaki et al., 1995), naphthomycin A

(Lu and Shen, 2007)—and two synthetic lactams (indolin-2-one

derivates HC-14 and HC-52; see Supplemental Data) were

used as sugar acceptors at 8.33 mM in standard assays with

UDP-Glc. Rifamycin A, rifampicin, and naphthomycin A were

not converted to the corresponding glycosides (data not shown).

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Amide N-Glycosylation

Figure 5. Substrate Specificity of Asm25 for Different Ansamitocin-Derived Aglycones and Sugar Donors

(A) ESI-MS spectra of the reaction products of Asm25 with different ansamitocin-derived aglycones. Panels 1–4: Incubations with different PNDs. Panel 5:

Incubation with equimolar mixture of four PNDs. All reactions were carried out in standard assays, at least in duplicate (see Supplemental Data).

(B) ESI-MS spectra of the reaction products of Asm25 with different UDP-sugar donors. For assaying different sugar donors, 10 ml of sugar nucleotide (0.15 mM

final concentration) and 10 ml PND-3 (0.04 mM final concentration) were added to 0.1 ml basal reaction solution for a 0.12 ml final volume. The reactions were

terminated after 2 h at 37�C and the products were detected by LC-MS. Each assay was carried out at least in duplicate.

However, lebstatin, 17-DMLB, HC-14, and HC-52 were glycosy-

lated by Asm25. In the LC-MS spectra, quasimolecular ion peaks

were seen for lebstatin at m/z 571.4 [M + Na]+ and for the ex-

868 Chemistry & Biology 15, 863–874, August 25, 2008 ª2008 Elsev

pected glycosylated product at m/z 733.4 [M + Na]+. Similarly,

in the LC-MS spectra of the incubation with 17-DMLB, the glyco-

sylated product was detected at m/z 719.4 [M + Na]+. Moreover,

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Chemistry & Biology

Amide N-Glycosylation

Figure 6. ESI-MS Spectra of the Glycosylation on Selected Nonansamitocin Aglycones by Asm25

17-DMLB, lebstatin, HC-14, and HC-52 were used as sugar acceptors at 8.33 mM in standard assays with UDP-Glc, respectively (see Supplemental Data).

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Chemistry & Biology

Amide N-Glycosylation

the corresponding glycosylated products of HC-14 and HC-52

were observed at m/z 366.5 [M + H]+ and 582.9 [M + Na]+,

respectively (Figure 6).

Antifungal and Antitumor Activitiesof PND-3, AP-3, and AGP-3AP-3, AGP-3, and PND-3 were tested for antifungal activity

against yeast Filobasidium uniguttulatum IFO 0699. AP-3 and

PND-3 showed remarkable activity against F. uniguttulatum at

50–200 ng/disc, but AGP-3 showed similar activity only at higher

concentration (8–10 mg/disc; Figure 7A). This assay indicated

that AP-3 possesses the strongest antifungal activity, followed

by PND-3 and AGP-3.

The antitumor activities of AP-3, AGP-3, and PND-3 against

the HepG2 cell line were assayed by the MTT method (Mos-

mann, 1983). AP-3 and PND-3 showed similar activities with

IC50 of 0.2 mg/ml and 0.1 mg/ml, respectively, which is consistent

with values reported for maytansinoids (Sneden and Beemster-

boer, 1980). AGP-3 was 10 times less active than AP-3

(Figure 7B). However, the in vivo antitumor activity of AGP-3

would still be worth testing because some maytansinoids have

been reported to have low activity during in vitro test systems

but a dramatic in vivo antitumor activity (Cassady et al., 2004).

DISCUSSION

The post-PKS modification reactions in the biosynthesis of ansa-

mitocins are diverse, including chlorination, carbamoylation,

O-methylation, acylation, epoxidation, and N-methylation. Nev-

ertheless, the structural variations among the naturally occurring

Figure 7. Antifungal and Antitumor Activi-

ties of AP-3, PND-3, and AGP-3

(A) Antifungal activity against F. uniguttulatum IFO

0699 of AP-3, PND-3, and AGP-3.

(B) The antitumor activities of AP-3, PND-3, and

AGP-3 against HepG2 cells. The IC50 of AP-3,

PND-3, and AGP-3 were calculated to be 0.2,

0.1, and 2.0 mg/ml, respectively.

ansamitocins are limited mainly to the

C-3 acyl moieties and to the omission of

one or two of the post-PKS modification

steps. Our isolation from A. pretiosum of

three novel ansamitocin amide N-glyco-

sides, AGP-1, AGP-2, and AGP-3

(Figure 1A), revealed a novel post-PKS

modification reaction not heretofore

encountered in ansamitocin biosynthesis

(Lu et al., 2004; Ma et al., 2007).

Bioinformatic analysis of the asm gene

cluster had revealed the presence of

asm25 encoding a putative Gtf, but its

function was obscure (Yu et al., 2002).

Asm25 showed highest sequence simi-

larity to CalG1 in the calicheamicin

biosynthetic gene cluster cloned from

Micromonospora echinospora ssp. cali-

chensis (27% identity, 42% similarity)

(Ahlert et al., 2002). In the present work, the Asm25 protein has

now been characterized as a dedicated tailoring Gtf for ansami-

tocins. Gtfs (EC 2.4.x.y) constitute a large family of enzymes that

are involved in the biosynthesis of oligosaccharides, polysac-

charides, glycoconjugates, and other natural products. Glyco-

syltransfers are nucleophilic replacement reactions between

C-1 of nucleotide-activated sugars and aglycones that usually

carry nucleophilic hydroxyl substituents. However, less fre-

quently, amines and nucleophilic carbons can also serve as

sugar acceptors, such as in plant xenobiotic metabolism by

the bifunctional O- and N-Gtf UGT72B1 (Brazier-Hicks et al.,

2007), and in the biosynthesis of antibiotics—e.g., rebeccamycin

(Sanchez et al., 2002), urdamycin (Hoffmeister et al., 2000), and

enterobactin (Fischbach et al., 2005). Many N-glycosides are

found among the indolocarbazole antibiotics (Sanchez et al.,

2006). The first N-Gtf-encoding gene (ngt/rebG) was cloned

from Lechevalieria aerocolonigenes ATCC 39243 (Hyun et al.,

2003; Nishizawa et al., 2005; Ohuchi et al., 2000; Onaka et al.,

2003a, 2003b; Sanchez et al., 2002). It is involved in the biosyn-

thesis of the indolocarbazole alkaloid rebeccamycin. Later, three

more N-Gtf genes, staG (Onaka et al., 2002; Salas et al., 2005),

atmG (Gao et al., 2006), and inkG (Kim et al., 2007), were

identified.

So far, the biochemical characteristics and/or substrate spec-

ificity profiles of the four known N-Gtfs involved in N-glycosyla-

tions of indolocarbazole antibiotics have been investigated

only by in vivo experiments due to the difficulty of obtaining sol-

uble active proteins (Sanchez et al., 2005; Zhang et al., 2006).

The expression of the rebG gene in E. coli and in Streptomyces

lividans led to RebG overproduction as inclusion bodies. All

870 Chemistry & Biology 15, 863–874, August 25, 2008 ª2008 Elsevier Ltd All rights reserved

Chemistry & Biology

Amide N-Glycosylation

efforts to obtain soluble and functional RebG were unsuccessful.

As the best outcome, RebG was partially solublized by fusion

with maltose binding protein or by coexpression with DnaK/

DnaJ chaperones; however, this soluble RebG was inactive dur-

ing in vitro assays (Zhang et al., 2006). Just as encountered in the

heterologous expression of RebG, StaG, and AtmG, the overpro-

duction of Asm25 in E. coli was successful, but produced inclu-

sion bodies. Because in the previous work many strategies had

been attempted without any success (Zhang et al., 2006), we

decided to focus on refolding Asm25 after purification from the

inclusion bodies under denaturing conditions, as this seemed

the most straightforward approach.

Our extensive efforts in refolding Asm25 from denaturated

protein eventually produced soluble active enzyme in reasonable

yield (�4.38 mg/l of induced cells). When inclusion bodies were

purified, 2 M urea was added in renaturation buffer IIa to further

purify Asm25 before solubilizing, and which also removed non-

specifically adsorbed proteins. To refold Asm25, we used a pro-

cedure similar to that described previously (Yang et al., 2004),

except that glycerol, which has long been known to increase pro-

tein stability (Gekko and Timasheff, 1981), and glycine were

added to renaturation buffer IV. After adding 0.1 M glycine, the

activity of Asm25 was increased dramatically (data not shown).

The use of low molecular-weight additives such as L-arginine,

sugars, glycerol, and sarcosine during the refolding process

often helps in improving the yield of active proteins from inclu-

sion bodies (Clark, 1998). These additives influence both the sol-

ubility and stability of the unfolded protein, folding intermediates,

and the fully folded protein. With the soluble Asm25 available, we

characterized the N-glycosylation reaction in vitro.

As anticipated, we found that UDP-Glc is the sugar donor and

PNDs are the sugar acceptors for the Asm25-catalyzed glycosyl-

ation to generate AGPs (Figure 1A). Using UDP-Glc and PND-3

as cosubstrates, we investigated the kinetics of renatured

Asm25 and found that the enzyme catalyzes the N-glycosylation

of PND-3 via the formation of a ternary complex (Marangoni,

2003; Price and Stevens, 1982). Ternary complex formation is

entirely in agreement with the theory—proposed on the basis

of structural and sequence homology studies of Gtfs—that

UDP-Glc transferases possess two major functional domains

(Mackenzie, 1990). Each of these domains is thought to contain

a binding site for one of the cosubstrates, enabling both sub-

strate molecules to bind to the enzyme simultaneously. This ter-

nary complex sequential mechanism is in agreement with the

limited studies carried out on other Gtfs (Jackson et al., 2001;

Mulichak et al., 2001).

The evaluation of the substrate specificity of Asm25 for various

sugar acceptors showed that all four PNDs tested (Figure 1A) are

used as substrates, but that the structure of the C-3 acyl group

modulates the efficiency of conversion. Both in individual incu-

bations and in a competition experiment with all four com-

pounds, PND-2 was found to be the preferred substrate. This

observation suggests that the C-3 acyl group may serve as

a binding site in a hydrophobic interaction between PNDs and

the aglycone binding pocket of Asm25. Additionally, this result

is consistent with the fact that AGP-2 was the most abundant

product in the fermentation of A. pretiosum on solid ISP2

medium (data not shown). Two N-desmethylansamitocins lack-

ing the 4,5-epoxy function, DDM and DDP-1 (Figure S1), were

Chemistry & Biology 15, 8

extremely poor substrates, if at all. This further supports the

speculation that the macrolactam amide N-methylation and/or

N-glycosylation is the final step in the post-PKS modifications

of ansamitocins (Spiteller et al., 2003). However, it is rather sur-

prising given the finding that two other benzenic ansamycins, the

geldanamycin derivatives lebstatin and 17-DMLB (Figure 5A), do

serve as substrates. The site of glycosylation has not been deter-

mined with these substrates, but is assumed to be the macrolac-

tam amide nitrogen. Three naphthalenic ansamycins, rifamycin

A, rifampicin, and naphthomycin A, however, were not glycosy-

lated by Asm25, although two synthetic lactams, HC-14 and

HC-52 (Figure 6), were converted to glycosides with modest

efficiency. Though these results do not provide a clear picture

of the aglycone binding site, they do indicate considerable pro-

miscuity of the enzyme toward sugar acceptor substrates.

The relative promiscuity of Asm25 with respect to the aglycone

substrate contrasts with the rather high specificity of the enzyme

for just UDP-Glc as the sugar donor. The UDP derivatives of

galactose, glucuronic acid, and N-acetylglucosamine, and

ADP- and GDP-Glc, were not used as substrates. Similarly, it

has been reported that no sugar moieties other than D-glucose

are utilized for the glycosylations catalyzed by RebG (Onaka

et al., 2003a, 2003b; Sanchez et al., 2002, 2005; Zhang et al.,

2006). Conversely, StaG accepts a variety of sugar moieties, in-

cluding L-rhamnose, L-olivose, L-digitoxose, and D-olivose dur-

ing in vivo biotransformations (Salas et al., 2005). An examination

of the structures in the PDB database shows that the conforma-

tions of UDP-hexose substrates bound to enzymes can vary sig-

nificantly. The variations occur largely around the diphosphate

linkage, which has shallow rotational barriers and the ability to

adopt a large number of isoenergetic conformations. Thus, the

nucleoside and hexose portions of the substrate can be pre-

sented in different orientations depending on the enzyme,

reflecting large differences in the substrate binding pockets.

SIGNIFICANCE

Ansamitocins are the microbial versions of natural maytan-

sinoids, a family of 19-membered macrocyclic lactams

with extraordinary cytotoxic and antineoplastic activities,

which are currently undergoing clinical evaluation as anti-

body-conjugated antitumor drugs (Widdison et al., 2006).

Their biosynthesis, catalyzed by the products of the asm bio-

synthetic gene cluster, involves the assembly of an initial

polyketide macrolactam, followed by a series of post-PKS

modifications introducing a chlorine, two methyl groups,

a cyclic carbamate, an ester side chain, and an epoxide

function to form the ansamitocins. The unexpected isolation

of ansamitocinosides from A. pretiosum cultivated on solid

ISP2 medium implies the further potentials for post-PKS

modifications of maytansinoids and calls for alternative

strategies for natural product mining. The identification of

Asm25 as the N-Gtf adding glucose moiety to the macrolac-

tam amide nitrogen enriches the antibiotic glycotransferase,

especially the N-Gtf, toolbox. Notably, heterologous expres-

sion of asm25 in E. coli and solubilization of the Asm25

protein from the inclusion bodies provided for the first time

a recombinant antibiotic N-glycosyltransferase in soluble,

enzymatically active form. This allowed the in vitro

63–874, August 25, 2008 ª2008 Elsevier Ltd All rights reserved 871

Chemistry & Biology

Amide N-Glycosylation

characterization of Asm25 to examine the properties of this

enzyme and the catalytic mechanism of this unusual C-N

bond forming glycosylation. The broad substrate range of

the enzyme for different aglycones suggests the possibility

of using it to generate N-glucosylated derivatives of ansa-

mycins and other compounds for evaluation as improved

bioactive agents or potential prodrugs.

EXPERIMENTAL PROCEDURES

Materials, General Methods, and Instrumentation

Optical rotations were measured with a JASCO DIP-370 digital polarimeter in

MeOH solution. Mass spectra were measured on an API Qstar Pulsar spec-

trometer. NMR spectra were recorded on Bruker AM-400 and DRX-500

NMR spectrometers with TMS as internal standard. UDP-Glc, rifamycin, and

rifampicin were purchased from Sigma-Aldrich. Naphthomycin A was isolated

from the endophytic Streptomyces sp. CS (Lu and Shen, 2007). The prepara-

tion of PND-1-4, DDM, DDP-1, lebstatin, 17-DMLB, HC-14, and HC-52 is

described in the Supplemental Data. UDP-galactose, UDP-N-acetyl-glucos-

amine, UDP-glucuronic acid, ADP-Glc, and GDP-Glc were obtained as a gift

from Taifo Mahmud of the Department of Pharmaceutical Sciences, Oregon

State University, Corvallis. LC-MS for qualitative and quantitative analyses

was carried out using a Waters series HPLC 2695 instrument (Waters Corp.)

with a Thermo Finnigan LCQ Advantage (Thermo Finnigan) electrospray ioni-

zation mass detector (ion trap). For methods of LC-MS, see Supplemental

Data.

Strains, Plasmids, Culture Techniques, and Media

Actinosynnema pretiosum ssp. aurantium ATCC 31565 and its derivatives, and

Streptomyces hygroscopicus XM201 were grown either on solid or in liquid

ISP2 medium (containing 0.4% yeast extract, 1% malt extract, and 0.4%

glucose [pH 7.3]) or liquid TSBY medium [30 g/l tryptic soy broth (LabM; Topley

House), 10 g/l yeast extract, and 103 g/l sucrose]. Fermentations were carried

out for 7 days at 28�C. Subsequent extractions and purifications were

performed as described in the Supplemental Data. E. coli DH10B (Invitrogen),

ET12567(pUZ8002) (MacNeil et al., 1992), and BL21(DE3)pLysE (Invitrogen)

were used as hosts for plasmid construction, E. coli-Actinosynnema biparental

conjugation, and protein overexpression, respectively. The yeast Filobasidium

unigutulatum IFO0699 was used as indicator strain for ansamitocin and ansa-

mitocinoside bioassay, and cytotoxicity assays were performed on a panel of

HepG2 cells. pBluescript KS(�) and pET26a were used for plasmid construc-

tion. pHGF9053 (Minagawa et al., 2007) was the vector used for the gene in-

activation of asm25 and pOJ2600, which is a derivative of pOJ260 (Bierman

et al., 1992) with the original KpnI site removed, was used for asm10 inactiva-

tion. Integrative plasmid pIB139 (Wilkinson et al., 2002) was used for the com-

plementation of the asm25 mutant. pRSET-B (Invitrogen) was used as vector

for protein overexpression in E. coli. For the complete list of strains and

plasmids used in this article, see Table S5.

Isolation and Identification of Ansamitocinosides

The strain A. pretiosum ATCC 31565 was inoculated on a slope of solid ISP2

medium in a test tube and cultivated for 5 days at 28�C to allow seed culture.

Solid-state fermentation was performed with solid ISP2 medium (3 L, �150

petri dishes) for 7 days at 28�C. The culture was extracted five times with

EtOAc-MeOH-AcOH (80:15:5, v/v/v) to afford a crude extract of 21.0 g. By

following the isolation procedure described previously (Ma et al., 2007), three

ansamitocin glucosides, AGP-1 (5.0 mg), AGP-2 (8.0 mg), and AGP-3 (3.0 mg),

were obtained. The NMR and MS data of AGP-1 and AGP-2 were identical with

the literature (Ma et al., 2007). The ESI-MS and NMR (1H, 13C, DEPT, HSQC

and HMBC) spectra (Figures S2A–L), HPLC comparison profile (Figure S2M),

and NMR assignments (Tables S1A and S1B) for in vivo and in vitro AGP-3

are given in the Supplemental Data.

Inactivation of Glycosyltransferase Gene asm25

A 6646 bp SacI fragment containing asm25 was cleaved from cosmid 19F11

(Yu et al., 2002) of the ATCC 31565 genomic library and cloned into pBluescript

KS(�) digested with SacI, resulting in pJTU476. The second plasmid,

872 Chemistry & Biology 15, 863–874, August 25, 2008 ª2008 Elsev

pJTU477, was constructed through the ligation of the 2742 bp SacI-PvuII

and 3439 bp PvuII-SacI fragments of pJTU476 with SacI-digested pET26a.

Transfer of the 6181 bp SacI fragment from pJTU477 to SacI-digested

pHGF9053 generated pJTU478 for the subsequent inactivation of asm25.

pJTU478 was introduced into ATCC 31565 via E. coli-Actinosynnema biparen-

tal conjugation as described (Kieser et al., 2000). Exconjugants were selected

with 15 mg/ml thiostrepton. For the screening for double-crossover mutants,

an exconjugant was inoculated into liquid TSBY medium and cultivated for

three rounds without the presence of thiostrepton. Then the thiostrepton-sen-

sitive derivatives were analyzed through PCR amplification with a pair of

primers (Asm25-Det-F: 50-CCCCAGCACGGAGGAAGA-30 and Asm25-Det-

R: 50-AGCGGAGGAGGAGACCCA-30). The PCR amplification was done in

a thermocycler (Thermo, MBS Satellite 0.2) under the following conditions:

35 cycles of 30 s at 94�C, 30 s at 50.5�C, and 90 s at 72�C. The asm25 mutant,

named BLQ11, was also analyzed by fermentation and LC-MS analysis.

Complementation of Mutant BLQ11 with Cloned asm25

A 1.55 kb DNA fragment containing the asm25 gene was cleaved from

pJTU812 by NdeI and EcoRI and cloned into plasmid pIB139 to obtain

pJTU813, which was introduced from E. coli into BLQ11 through conjugation.

Confirmation was carried out through plasmid isolation from the exconjugants,

plasmid transformation of E. coli, and comparison between the newly purified

pJTU813 and the original one by restriction enzyme digestions. An exconju-

gant carrying the correct plasmid was named as BLQ15 and characterized

by fermentation and LC-MS analysis.

Cloning and Heterologous Expression of Recombinant

His6-tagged Asm25

Gene asm25 was amplified from the ATCC 31565 genome by PCR using

primers asm25-F (50-GGATCCACATATGCGCGTTCTGTTCACC-30, engi-

neered BamHI and NdeI sites underlined) and asm25-R (50-GAATTCACAC

CACCGCGACGCTC-30, engineered EcoRI site underlined). The amplified

gene fragment was digested with BamHI and EcoRI and ligated with

pRSET-B digested with the same enzymes to generate pJTU812. The se-

quence of the cloned asm25 was confirmed by DNA sequencing. pJTU812

was transformed into E. coli BL21(DE3)pLysE, and transformants were grown

in LB medium supplemented with ampicillin (100 mg/ml) and chloramphenicol

(17.5 mg/ml) at 37�C and 230 rpm for 12 h, then diluted 1:10 with fresh LB me-

dium. The diluted cultures were grown to OD600 = 0.4�0.6, then induced with

0.1�0.8 mM IPTG and incubated for 5 h at 37�C, 230 rpm.

Purification and Renaturation of Asm25 from Inclusion Bodies

E. coli cells (200 ml) induced for 5 h at 37�C were centrifuged at 8,000 g for

15 min and the pellet was resuspended in 3 ml renaturation buffer I (50 mM

Tris-HCl [pH 8.0], 300 mM NaCl, 10 mM MgCl2). Cells were then frozen in liquid

nitrogen, rapidly thawed in 37�C water for three cycles, and then sonicated on

ice six times for 15 s each at 15 s intervals. After centrifugation of the lysate at

4�C and 30,000 g for 20 min, the inclusion body pellet was resuspended in 3.0

ml renaturation buffer IIa (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10 mM

MgCl2, 0.5% Triton, 2.0 M urea) for 5 min at room temperature and recovered

by centrifugation at 4�C and 15,000 g for 15 min. The same procedure was re-

peated twice in 3.0 ml renaturation buffer IIb (50 mM Tris-HCl [pH 8.0], 300 mM

NaCl, 10 mM MgCl2, 0.5% Triton). The purified Asm25 inclusion bodies were

suspended in 4.0 ml renaturation buffer III (50 mM Tris-HCl [pH 8.0], 300 mM

NaCl, 10 mM MgCl2, 8.0 M urea) and centrifuged at 8,000 g for 15 min; the pel-

let was then discarded. The supernatant was dialyzed against renaturation

buffer IV (50 mM Tris-HCl [pH 8.0], 10% glycerol, 100 mM NaCl, 0.1 M glycine,

0.5 mM EDTA, 1.0 mM DDT, 10 mM MgCl2) for 24 h at 4�C. After centrifugation

for 15 min at 4�C and 20,000 g, the supernatant (12.0 ml) was collected and

used for subsequent enzyme assays. Protein purity and molecular mass

were examined by SDS-PAGE followed by Coomassie Brilliant Blue staining

of the gel. For SDS-PAGE, the protein solution was mixed with Laemmli sam-

ple buffer containing b-mercaptoethanol (5%) and incubated at 40�C for

30 min. The protein concentration was measured to be 1.6 mM by the Bradford

method or by staining intensity compared with BSA concentration standards.

The yield of Asm25 was calculated to be�4.38 mg/l of induced cells. To make

a basal reaction solution for activity assay and storage at 4�C, glycerol and

ier Ltd All rights reserved

Chemistry & Biology

Amide N-Glycosylation

BSA were added to the enzyme solution in buffer IV at final concentrations of

30% and 1%, respectively.

SUPPLEMENTAL DATA

Supplemental Data include five figures, five tables, Supplemental Experimen-

tal Procedures, and Supplemental References, and can be found with this

article online at http://www.chembiol.com/cgi/content/full/15/8/863/DC1/.

ACKNOWLEDGMENTS

We thank Taifo Mahmud at Oregon State University, Corvallis, for providing

UDP-galactose, UDP-N-acetyl-glucosamine, UDP-glucuronic acid, ADP-Glc,

and GDP-Glc. This work was partially supported by the National Natural

Science Fund for Distinguished Young Scholars to Y. Shen (30325044), the

Natural Science Foundation of China (30600005, 30570019), and the Ministry

of Science and Technology (973 and 863 Programs).

Received: February 14, 2008

Revised: June 10, 2008

Accepted: June 13, 2008

Published: August 22, 2008

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